Stimulation of the anaerobic growth ofSalmonella typhimurium by reduction ofl-carnitine,carnitine derivatives and structure-related trimethylammonium compounds

In view of the development of al-carnitine deficiency, the metabolism ofl-carnitine and structure-related trimethylammonium compounds was studied inSalmonella typhimurium LT₂ by means of thin-layer chromatography (TLC).l-Carnitine, crotonobetaine and acetyl-l-carnitine stimulated the anaerobic growth in a complex medium significantly. The stimulation depended on the formation of -butyrobetaine. The reduction ofl-carnitine proceeded in two steps: (1) Dehydration of thel-carnitine to crotonobetaine, (2) hydrogenation of crotonobetaine to -butyrobetaine. The reduction of crotonobetaine was responsible for the growth stimulation. Terminal electron acceptors of the anaerobic respiration such as nitrate and trimethylamine N-oxide, but not fumarate, suppressed the catabolism ofl-carnitine completely. Glucose fermentation, too, inhibited the reduction ofl-carnitine but optimal growth with a high carnitine catabolism was achieved byd-ribose. The esters of carnitine with medium- and long-chain fatty acids inhibited the growth considerably because of their detergent properties.Abbreviations TLC thin-layer chromatography     

Respiration-driven proton translocation with nitrite and nitrous oxide in Paracoccus denitrificans

(1) $\text{H}$⁺/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O₂, NO^?₂ or N₂O. Under optimal H⁺-translocation conditions, the ratios $\text{H}{}^{\mathrm{+}}\text{O}$, $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO^?₂ or N₂O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were ?0.84, ?2.33 and ?1.90, respectively. (3) The $\text{H}{}^{\mathrm{+}}\text{oxidant}$ ratios, mentioned in item 2, were not altered in the presence of $\text{valinomycin}\text{K}{}^{\mathrm{+}}$ and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O₂, NO^?₂ and N₂O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO^?₂, N₂O or O₂ pass two H⁺-translocating sites. The $\text{H}{}^{\mathrm{+}}\text{electron}$ acceptor ratios predicted by this scheme are in good agreement with the experimental values.     

Role of sugars in nitrate utilization by roots of dwarf bean

Nitrate uptake and in vivo, nitrate reductase activity (NRA) in roots of Phaseolus vulgaris, L. cv. Witte Krombek were measured in nitrogen-depleted plants of varying sugar status, Variation in sugar status was achieved at the start of nitrate nutrition by excision, ringing, darkness or administration of sugars to the root medium. The shape of the apparent induction pattern of nitrate uptake was not influenced by the sugar status of the absorbing tissue. When measured after 6 h of nitrate nutrition (0.1 mol m^?3), steady state nitrate uptake and root NRA were in the order intact>dark>ringed>excised. Exogenous sucrose restored NRA in excised roots to the level of intact plants. The nitrate uptake rate of excised roots, however, was not fully restored by sucrose (0.03–300 mol m^?3). When plants were decapitated after an 18 h NO₃^? pretreatment, the net uptake rate declined gradually to become negative after three hours. This decline was slowed down by exogenous fructose, whilst glucose rapidly (sometimes within 5 min) stimulated NG^?₃ uptake. Presumably due to a difference in NO₃^? due to a difference in NO₃^? uptake, the NRA of excised roots was also higher in the presence of glucose than in the presence of fructose after 6 h of nitrate nutrition. The sugar-stimulation of, oxygen consumption as well as the release of ¹⁴CO₂ from freshly absorbed (U-¹⁴C) sugar was the same for glucose and fructose. Therefore, we propose a glucose-specific effect on NO₃^? uptake that is due to the presence of glucose rather than to its utilization in root respiration. A differential glucose-fructose effect on nitrate reductase activity independent of the effect on NO₃^? uptake was not indicated. A constant level of NRA occurred in roots of NO₃^? induced plants. Removal of nutrient nitrate from these plants caused an exponential NRA decay with an approximate half-life of 12 h in intact plants and 5.5 h in excised roots. The latter value was also found in roots that were excised in the presence of nitrate, indicating that the sugar status primarily determines the apparent rate of nitrate reductase decay in excised roots.     

Pigment distribution and nitrogen fixation inAnabaena azollae

Summary The symbiotic heterocystous cyanobacteriumAnabaena azollae present in the leaf cavities of the water fernAzolla spp. was studied. The cyanobacteria extracted from the leaf cavities showed differences in pigment composition in three species ofAzolla, i.e A.pinnata var.pinnata, A.caroliniana and A.filiculoides, as observed by pigment absorption and epifluorescence tests. These differences suggest that of these species the cyanobiont ofA. pinnata is the most actively nitrogenfixing form. This has been confirmed by nitrogen fixation (acetylene reduction) tests. Heterocysts of the symbiont ofA. pinnata were characterized by high chlorophylla and low phycocyanin content, a low fluorescence yield of chlorophyll in the heterocysts compared to vegetative cells and a gradient of phycocyanin concentration in the vegetative cells adjacent to heterocysts. This indicates that only photosystem I is present in the heterocyst. In the two otherAzolla species quantitative shifts in the pigment composition occurred suggesting a lower nitrogen fixation activity.In the cyanobiontAnabaena azollae the heterocyst frequency could reach a value of 44–45%. It is argued that there are two generations of heterocysts in a matureAzolla plant, which are concomitant with two peaks of nitrogen fixation activity correlated with leaf age,i.e. leaf number along the main axis of the plant. At both peaks of maximal N₂-ase activity, only 20–25% of the heterocysts present are metabolically active as demonstrated by the reduction of Neotetrazolium chloride (NTC) in the heterocysts and darkening of nuclear emulsions by silver salt reduction. Vegetative cells of the cyanobiont reduce Neotetrazolium chloride (NTC) to formazan more rapidly than has been observed in the free-living heterocystous cyanobacteriumAnabaena cylindrica tested in parallel experiments. This feature may be due to a more permeable cell wall of the vegetative cells of the cyanobiont compared to the free-living form, since the vegetative cells of the symbiont play a role in cross-feeding of the host (Azolla).Evidence is obtained that only the heterocysts of the cyanobiont ofAzolla are involved in the nitrogen fixation process as in free-living heterocystous cyanobacterium species. This situation is different from other cyanobacterial symbioses such as inGunnera, Blasia andAnthoceros, where physiological modifications are reported in the symbiosis with another photosynthetic partner such as the absence of O₂ evolution and the absence of photo-fixation of CO₂ in the cyanobionts.Pigment composition and N₂-ase activity in the symbiotic cyanobacteria of three Azolla species have indicated the superiority of theA. pinnata symbiont.A. pinnata var.pinnata is a semidomesticated form used in S.E. Asia for agricultural purposes (irrigated rice culture) to increase soil fertility.It is suggested that by selection (domestication) more efficient strains (clones) can be obtained, and further that with more advanced techniques such as gene mutation and genetic manipulation even more efficient and for agriculture more beneficial clones can be obtained.     

Proteoid root morphology and function inLupinus albus

Summary Current theories of phosphorus uptake by plants imply that they can augment diffusion to their root axes by the development of abundant root hairs or mycorrhizas. Some phosphorus efficient plants have root morphology with multi-branched roots and localised regions of densely packed root hairs, which we suggest is better suited to the retention of substances exuded by the roots than uptake of substances moving to the root by diffusion. Evidence of substantial exudation by the proteoid roots ofLupinus albus is presented.     

The effect of the osmotic potential and specific ion concentration of the nutrient solution on the uptake and reduction of nitrate by barley seedlings

Summary Short-term absorption experiments were conducted with intact barley (Hordeum vulgare L.) seedlings to observe the effects of the osmotic potential (Ψ^π) and salt species on nitrate uptake andin vivo nitrate reduction. The experiments consisted of growing barley seedlings for 5 days in complete nutrient solutions salinized to (Ψ^π) levels of −0.6, −1.8, −3.0, −4.2, and −5.4 bars with NaCl, CaCl₂ or Na₂SO₄. After the absorption period, the seedlings were separated into shoots and roots, weighed, then analyzed for NO₃. The nutrient solutions were sampled for NO₃ analysis each day immediately before renewing the solutions. The accumulative loss of NO₃ from the solutions was considered to be uptake whereas NO₃ reduction was the difference between uptake and seedling content. Lowering the (Ψ^π) of the nutrient solutions resulted in decreased concentrations of NO₃ in the plant, little or no effect (except at the lowest (Ψ^π) level) on uptake, and increased nitrate reductase activity. Increased rates of NO₃ reduction were in particular associated with the Cl concentration of the nutrient solution.     

DISTRIBUTION AND PHYSIOLOGICAL DETERMINANTS OF BLUE-GREEN ALGAL NITROGEN FIXATION ALONG A THERMOGRADIENT1

The capacity of thermal algal-bacterial mats to fix nitrogen (N₂) was examined in an alkaline thermal stream, Rabbit Creek, of Yellowstone National Park. Nitrogenase activity and nitrogen-fixation rates of mat cores placed in serum bottles and incubated in situ were estimated by the acetylene-reduction technique. Active nitrogenase was not detected at 60 or 65 C in either the blue-green algal or bacterial undermat components of the mats. Acetylene was reduced by all mats ≤55 C along the thermogradient; mean fixation estimates for the mats ranged from 7 to 5,028 nmoles N₂ fixed · mg Chl a^?1· hr^?1. Maximum fixation occurred at 35 C in the stream; statistical comparison of mean rates ordered the thermogradient mats according to estimated activities: 35 > 40 > 30 > 50 ≥ 55 ≥ 45 C. Mats (≤40 C) dominated by species of Calothrix accounted for ca. 97% of the total nitrogen fixation observed in the stream; the remaining activity was associated with mats containing Mastigocladus laminosus Cohn. Light intensity significantly affected fixation rates of the Calothrix mats which responded in a linear fashion from 9–100% full sunlight (ca. 1,900 μEin · m^?2· sec^?1). Calothrix mats from 30 and 40 C had maximum nitrogenase activity at their growth temperature suggesting that nitrogen fixation along the thermogradient was optimally adapted to in situ temperatures.     

Maintenance of the Adult Rat Superior Cervical Ganglion In Vitro: Comparison of Organ and Explant Culture Systems

Abstract: Concentrations of selected intermediates of energy metabolism whole rat superior cervical ganglia maintained in vitro by an organ culture technique were compared with values measured in small slices of this maintained under essentially the same conditions. Rates of incorporation [³H]leucine into trichloroacetic acid-precipitable material in whole ganglia mained constant for at least 48 h: however, the oxidation-reduction state tissue as indexed by (NAD):(NADH) ratios calculated from measured amounts of lactate and pyruvate decreased more than 50% within ³h in vitro . Ganglion explants prepared by cutting the tissue into ³⁰⁰-pm transverse sections played (NAD):(NADH) ratios that were about three times greater than noted in whole ganglia maintained in vitro for the same period of time. explants contained significantly higher concentrations of pyruvate and α-ketoglutarate than whole ganglia maintained in culture. Maintenance of vorable metabolic state may support the extensive growth of neurites seen explant cultures of superior cervical ganglia. Outgrowth of processes containing catecholamines could be detected readily in explant cultures of ganglia adult rats; however, this was somewhat slower and less consistent than growth observed in explants from neonatal rats. Outgrowth of neurites adult ganglia was minimal without the addition of Nerve Growth Factor.     

Carbon and nitrogen metabolism in legume root nodules

The literature concerning the metabolism of carbon compounds during the reduction, assimilation and translocation of nitrogen in root nodules of leguminous plants is reviewed. The reduction of dinitrogen requires an energy source (ATP) and a reluctant which are both supplied by respiratory catabolism of carbohydrates produced by the host plant. Photosynthates are also required to generate the carbon skeletons for amino acid or urcide synthesis during the assimilation of ammonia produced by the bacteria within the nodule tissue. Competition for photosynthates occurs between the bacteroids, nodule tissue and the various vegetative and reproductive sinks in the host plant. The nature of carbon compounds involved in these processes, their routes of metabolism, the mechanisms of control and the partitioning of metabolises between the various sites of utilization are only poorly understood. It is apparent that dinitrogen is reduced to ammonia in the bacteroids. Both fast- and slow-growing strains of Rhizobium possess the Entner-Doudoroff pathway of glucose catabolism, and some, if not all, enzymes of the Emden-Meyerhof pathway. Some bacterial cultures also metabolize carbon through the ketogluconate pathway but only the fast-growing strains of cultured rhizobia possess the key enzyme of the pentose phosphate pathway (6-phosphogluconate dehydrogenase). The host cells are thought to contain the complete Emden-Meyerhof pathway and tricarboxylic acid cycle, which provides the carbon skeletons for assimilation of the ammonia, formed by the bacteroids, into α-amino acids. A pathway of anapleurotic carbon conservation, operative in the host cells, synthesizes oxaloacetic acid through β-carboxylation of phosphoenol pyruvate. This process could be important in the recapture and assimilation of respired CO₂ in the rhizosphere. The main route of assimilation of ammonia produced by the bacteroids would appear to be via the glutamine synthetase-glutamate synthase pathway in the host cells. However, glutamate dehydrogenase may also be involved in ammonia assimilation. These enzymes also occur in in vitro cultures of Rhizobium and in bacteroids where they presumably participate in the synthesis of amino acids for growth of the bacteria or bacteroids. Nitrogen assimilated into glutamine or glutamate is exported from the nodules in a variety of forms, which include asparagine, glutamine, aspartate, homoserine and allantoates, in proportions which depend on the legume species. Studies on regulation of the overall process have focussed on expression of bacteroid genes and on the control of enzyme activity, at the level of nitrogenase and enzymes of nitrogen assimilation in particular. However, due to the wide range of experimental techniques, environmental conditions and plant species which have been used, no clear conclusions can yet be drawn. The pathways of carbon flow in nitrogen metabolism, particularly in relation to the synthesis of ureides and the regulation of carbon metabolism, remain key areas for future research in symbiotic nitrogen fixation.     

Nitrogen fixation in hardwood forests of the northeastern United States

Summary Nitrogen-fixing activity in hardwood forests of the northeastern United States occurred in wood litter, greater than 2 cm in diameter. Activity in large dead wood was independent of species, in the case of deciduous wood litter, but was restricted to partially decayed wood with a high moisture content. Maximum rates of activity were observed in the summer months, minimum rates in the winter. Evidence from six stands of varying ages showed that fixation in large wood litter occurred in only 25% of the samples assayed.Fixation was highest in the youngest, 4 years, and oldest, over 200 years, stands; being about 2 kg/ha/yr. The quantity of nitrogen fixed appears to be related to the biomass of dead wood. Large amounts of wood litter in the youngest stands were from slash left after cutting. As the supply of slash is exhausted by decay, nitrogen fixation decreases, with a low around year 20. Fixation then gradually increases as natural thinning adds wood to the litter compartment.Apparently, the amount of nitrogen fixed in dead wood the first 20 years following clearcutting can only replace a modest fraction of the amount lost as a result of the cutting and product removal. Finally, the results indicate that nitrogen fixation in wood litter does not equal nitrogen fixation in a northern hardwood forest calculated using a mass balance approach, suggesting that additional nitrogen inputs exist.